Multiple Horizontal Mini-chromosome Transfers Drive Genome Evolution of Clonal Blast Fungus Lineages.

Crop disease pandemics are often driven by asexually reproducing clonal lineages of plant pathogens that reproduce asexually. How these clonal pathogens continuously adapt to their hosts despite harboring limited genetic variation, and in absence of sexual recombination remains elusive. Here, we reveal multiple instances of horizontal chromosome transfer within pandemic clonal lineages of the blast fungus Magnaporthe (Syn. Pyricularia) oryzae. We identified a horizontally transferred 1.2Mb accessory mini-chromosome which is remarkably conserved between M. oryzae isolates from both the rice blast fungus lineage and the lineage infecting Indian goosegrass (Eleusine indica), a wild grass that often grows in the proximity of cultivated cereal crops. Furthermore, we show that this mini-chromosome was horizontally acquired by clonal rice blast isolates through at least nine distinct transfer events over the past three centuries. These findings establish horizontal mini-chromosome transfer as a mechanism facilitating genetic exchange among different host-associated blast fungus lineages. We propose that blast fungus populations infecting wild grasses act as genetic reservoirs that drive genome evolution of pandemic clonal lineages that afflict cereal crops.

Evolution and related pathogenic genes of Pseudodiploöspora longispora on Morchella based on genomic characterization and comparative genomic analysis.

True morels (Morchella) are globally renowned medicinal and edible mushrooms. White mold disease caused by fungi is the main disease of Morchella, which has the characteristics of wide incidence and strong destructiveness. The disparities observed in the isolation rates of different pathogens indicate their varying degrees of host adaptability and competitive survival abilities. In order to elucidate its potential mechanism, this study, the pathogen of white mold disease from Dafang county, Guizhou Province was isolated and purified, identified as Pseudodiploöspora longispora by morphological, molecular biological and pathogenicity tests. Furthermore, high-quality genome of P. longisporus (40.846 Mb) was assembled N50 of 3.09 Mb, predicts 7381 protein-coding genes. Phylogenetic analysis of single-copy homologous genes showed that P. longispora and Zelopaecilomyces penicillatus have the closest evolutionary relationship, diverging into two branches approximately 50 (44.3-61.4) MYA. Additionally, compared with the other two pathogens causing Morchella disease, Z. penicillatus and Cladobotryum protrusum, it was found that they had similar proportions of carbohydrate enzyme types and encoded abundant cell wall degrading enzymes, such as chitinase and glucanase, indicating their important role in disease development. Moreover, the secondary metabolite gene clusters of P. longispora and Z. penicillatus show a high degree of similarity to leucinostatin A and leucinostatin B (peptaibols). Furthermore, a gene cluster with synthetic toxic substance Ochratoxin A was also identified in P. longispora and C. protrusum, indicating that they may pose a potential threat to food safety. This study provides valuable insights into the genome of P. longispora, contributing to pathogenicity research.

Hi-C2B: Optimized Detection of Chromosomal Contacts Within Synchronized Meiotic S. cerevisiae Cells.

Hi-C, a genome-wide chromosome conformation capture assay, is a powerful tool used to study three-dimensional genome organization by converting physical pairwise interactions into counts of pairwise interactions. To study the many temporally regulated facets of meiotic recombination in S. cerevisiae, the Hi-C assay must be robust such that fine- and wide-scale comparisons between genetic datasets can be made. Here we describe an updated protocol for Hi-C (Hi-C2B) that generates reproducible libraries of interaction data with low noise and for a relatively low cost.

Characteristics of Corynespora cassiicola, the causal agent of tobacco Corynespora leaf spot, revealed by genomic and metabolic phenomic analysis.

Corynespora cassiicola is a highly diverse fungal pathogen that commonly occurs in tropical, subtropical, and greenhouse environments worldwide. In this study, the isolates were identified as C. cassiicola, and the optimum growth and sporulation were studied. The phenotypic characteristics of C. cassiicola, concerning 950 different growth conditions, were tested using Biolog PM plates 1-10. In addition, the strain of C. cassiicola DWZ from tobacco hosts was sequenced for the using Illumina PE150 and Pacbio technologies. The host resistance of tobacco Yunyan 87 with different maturity levels was investigated. In addition, the resistance evaluation of 10 common tobacco varieties was investigated. The results showed that C. cassiicola metabolized 89.47% of the tested carbon source, 100% of the nitrogen source, 100% of the phosphorus source, and 97.14% of the sulfur source. It can adapt to a variety of different osmotic pressure and pH environments, and has good decarboxylase and deaminase activities. The optimum conditions for pathogen growth and sporulation were 25-30 °C, and the growth was better on AEA and OA medium. The total length of the genome was 45.9 Mbp, the GC content was 51.23%, and a total of 13,061 protein-coding genes, 202 non-coding RNAs and 2801 and repeat sequences were predicted. Mature leaves were more susceptible than proper mature and immature leaves, and the average diameter of diseased spots reached 17.74 mm at 12 days. None of the tested ten cultivars exhibited obvious resistance to Corynespora leaf spot of tobacco, whereby all disease spot diameters reached > 10 mm and > 30 mm when at 5 and 10 days after inoculation, respectively. The phenotypic characteristics, genomic analysis of C. cassiicola and the cultivar resistance assessment of this pathogen have increased our understanding of Corynespora leaf spot of tobacco.

ChEC-Seq: A Comprehensive Guide for Scalable and Cost-Efficient Genome-Wide Profiling in Saccharomyces cerevisiae.

Chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq) is a profiling method for protein-DNA interactions that can detect binding locations in vivo, does not require antibodies or fixation, and provides genome-wide coverage at near nucleotide resolution.The core of this method is an MNase fusion of the target protein, which allows it, when triggered by calcium exposure, to cut DNA at its binding sites and to generate small DNA fragments that can be readily separated from the rest of the genome and sequenced.Improvements since the original protocol have increased the ease, lowered the costs, and multiplied the throughput of this method to enable a scale and resolution of experiments not available with traditional methods such as ChIP-seq. This method describes each step from the initial creation and verification of the MNase-tagged yeast strains, over the ChEC MNase activation and small fragment purification procedure to the sequencing library preparation. It also briefly touches on the bioinformatic steps necessary to create meaningful genome-wide binding profiles.

Discovery of a Hybrid Molecule with Phytotoxic Activity by Genome Mining, Heterologous Expression, and OSMAC Strategy.

Genome mining in association with the OSMAC (one strain, many compounds) approach provides a feasible strategy to extend the chemical diversity and novelty of natural products. In this study, we identified the biosynthetic gene cluster (BGC) of restricticin, a promising antifungal agent featuring a reactive primary amine, from the fungus Aspergillus sclerotiorum LZDX-33-4 by genome mining. Combining heterologous expression and the OSMAC strategy resulted in the production of a new hybrid product (1), along with N-acetyl-restricticin (2) and restricticinol (3). The structure of 1 was determined by spectroscopic data, including optical rotation and electronic circular dichroism (ECD) calculations, for configurational assignment. Compound 1 represents a fusion of restricticin and phytotoxic cichorin. The biosynthetic pathway of 1 was proposed, in which the condensation of a primary amine of restricticin with a precursor of cichorine was postulated. Compound 1 at 5 mM concentration inhibited the growth of the shoots and roots of Lolium perenne, Festuca arundinacea, and Lactuca sativa with inhibitory rates of 71.3 and 88.7% for L. perenne, 79.4 and 73.0% for F. arundinacea, and 58.2 and 52.9% for L. sativa. In addition, compound 1 at 25 µg/mL showed moderate antifungal activity against Fusarium fujikuroi and Trichoderma harzianum with inhibition rates of 22.6 and 31.6%, respectively. These results suggest that heterologous expression in conjunction with the OSMAC approach provides a promising strategy to extend the metabolite novelty due to the incorporation of endogenous metabolites from the host strain with exogenous compounds, leading to the production of more complex compounds and the acquisition of new physiological functions.

Isolation, genome analysis and tissue localization of Ceratobasidium theobromae, a new encounter pathogen of cassava in Southeast Asia.

In Southeast Asia (SEA) fastidious fungi of the Ceratobasidium genus are associated with proliferation of sprouts and vascular necrosis in cacao and cassava, crops that were introduced from the tropical Americas to this region. Here, we report the isolation and in vitro culture of a Ceratobasidium sp. isolated from cassava with symptoms of witches' broom disease (CWBD), a devastating disease of this crop in SEA. The genome characterization using a hybrid assembly strategy identifies the fungus as an isolate of the species C. theobromae, the causal agent of vascular streak dieback of cacao in SEA. Both fungi have a genome size > 31 Mb (G+C content 49%), share > 98% nucleotide identity of the Internal Transcribed Spacer (ITS) and > 94% in genes used for species-level identification. Using RNAscope® we traced the pathogen and confirmed its irregular distribution in the xylem and epidermis along the cassava stem, which explains the obtention of healthy planting material from symptom-free parts of a diseased plant. These results are essential for understanding the epidemiology of CWBD, as a basis for disease management including measures to prevent further spread and minimize the risk of introducing C. theobromae via long-distance movement of cassava materials to Africa and the Americas.

Analysis of Whole-Genome for Identification of Seven Penicillium Species with Significant Economic Value.

The Penicillium genus exhibits a broad global distribution and holds substantial economic value in sectors including agriculture, industry, and medicine. Particularly in agriculture, Penicillium species significantly impact plants, causing diseases and contamination that adversely affect crop yields and quality. Timely detection of Penicillium species is crucial for controlling disease and preventing mycotoxins from entering the food chain. To tackle this issue, we implement a novel species identification approach called Analysis of whole GEnome (AGE). Here, we initially applied bioinformatics analysis to construct specific target sequence libraries from the whole genomes of seven Penicillium species with significant economic impact: P. canescens, P. citrinum, P. oxalicum, P. polonicum, P. paneum, P. rubens, and P. roqueforti. We successfully identified seven Penicillium species using the target we screened combined with Sanger sequencing and CRISPR-Cas12a technologies. Notably, based on CRISPR-Cas12a technology, AGE can achieve rapid and accurate identification of genomic DNA samples at a concentration as low as 0.01 ng/µL within 30 min. This method features high sensitivity and portability, making it suitable for on-site detection. This robust molecular approach provides precise fungal species identification with broad implications for agricultural control, industrial production, clinical diagnostics, and food safety.

Complete genome of the Medicago anthracnose fungus, Colletotrichum destructivum, reveals a mini-chromosome-like region within a core chromosome.

Colletotrichum destructivum (Cd) is a phytopathogenic fungus causing significant economic losses on forage legume crops (Medicago and Trifolium species) worldwide. To gain insights into the genetic basis of fungal virulence and host specificity, we sequenced the genome of an isolate from Medicago sativa using long-read (PacBio) technology. The resulting genome assembly has a total length of 51.7 Mb and comprises ten core chromosomes and two accessory chromosomes, all of which were sequenced from telomere to telomere. A total of 15, 631 gene models were predicted, including genes encoding potentially pathogenicity-related proteins such as candidate-secreted effectors (484), secondary metabolism key enzymes (110) and carbohydrate-active enzymes (619). Synteny analysis revealed extensive structural rearrangements in the genome of Cd relative to the closely related Brassicaceae pathogen, Colletotrichum higginsianum. In addition, a 1.2 Mb species-specific region was detected within the largest core chromosome of Cd that has all the characteristics of fungal accessory chromosomes (transposon-rich, gene-poor, distinct codon usage), providing evidence for exchange between these two genomic compartments. This region was also unique in having undergone extensive intra-chromosomal segmental duplications. Our findings provide insights into the evolution of accessory regions and possible mechanisms for generating genetic diversity in this asexual fungal pathogen.

Datasets of fungal diversity and pseudo-chromosomal genomes of mangrove rhizosphere soil in China.

With climate change and anthropic influence on the coastal ecosystems, mangrove ecosystems are disappearing at an alarming rate. Accordingly, it becomes important to track, study, record and store the mangrove microbial community considering their ecological importance and potential for biotechnological applications. Here, we provide information on mangrove fungal community composition and diversity in mangrove ecosystems with different plant species and from various locations differing in relation to anthropic influences. We describe twelve newly assembled genomes, including four chromosomal-level genomes of fungal isolates from the mangrove ecosystems coupled with functional annotations. We envisage that these data will be of value for future studies including comparative genome analysis and large-scale temporal and/or spatial research to elucidate the potential mechanisms by which mangrove fungal communities assemble and evolve. We further anticipate that the genomes represent valuable resources for bioprospecting related to industrial or clinical uses.

Draft genome sequence and annotation of the polyextremotolerant polyol lipid-producing fungus aureobasidium pullulans NRRL 62042.

OBJECTIVES: The ascomycotic yeast-like fungus Aureobasidium exhibits the natural ability to synthesize several secondary metabolites, like polymalic acid, pullulan, or polyol lipids, with potential biotechnological applications. Combined with its polyextremotolerance, these properties make Aureobasidium a promising production host candidate. Hence, plenty of genomes of Aureobasidia have been sequenced recently. Here, we provide the annotated draft genome sequence of the polyol lipid-producing strain A. pullulans NRRL 62042. DATA DESCRIPTION: The genome of A. pullulans NRRL 62042 was sequenced using Illumina NovaSeq 6000. Genome assembly revealed a genome size of 24.2 Mb divided into 39 scaffolds with a GC content of 50.1%. Genome annotation using Genemark v4.68 and GenDBE yielded 9,596 genes.

MCM2-7 loading-dependent ORC release ensures genome-wide origin licensing.

Origin recognition complex (ORC)-dependent loading of the replicative helicase MCM2-7 onto replication origins in G1-phase forms the basis of replication fork establishment in S-phase. However, how ORC and MCM2-7 facilitate genome-wide DNA licensing is not fully understood. Mapping the molecular footprints of budding yeast ORC and MCM2-7 genome-wide, we discovered that MCM2-7 loading is associated with ORC release from origins and redistribution to non-origin sites. Our bioinformatic analysis revealed that origins are compact units, where a single MCM2-7 double hexamer blocks repetitive loading through steric ORC binding site occlusion. Analyses of A-elements and an improved B2-element consensus motif uncovered that DNA shape, DNA flexibility, and the correct, face-to-face spacing of the two DNA elements are hallmarks of ORC-binding and efficient helicase loading sites. Thus, our work identified fundamental principles for MCM2-7 helicase loading that explain how origin licensing is realised across the genome.

Genome sequencing and CAZymes repertoire analysis of Diaporthe eres P3-1W causing postharvest fruit rot of 'Hongyang' kiwifruit in China.

Postharvest rot caused by various fungal pathogens is a damaging disease affecting kiwifruit production and quality, resulting in significant annual economic losses. This study focused on isolating the strain P3-1W, identified as Diaporthe eres, as the causal agent of 'Hongyang' postharvest rot disease in China. The investigation highlighted cell wall degrading enzymes (CWDEs) as crucial pathogenic factors. Specially, the enzymatic activities of cellulase, ß-galactosidase, polygalacturonase, and pectin methylesterases peaked significantly on the second day after infection of D. eres P3-1W. To gain a comprehensive understanding of these CWDEs, the genome of this strain was sequenced using PacBio and Illumina sequencing technologies. The analysis revealed that the genome of D. eres P3-1W spans 58,489,835 bp, with an N50 of 5,939,879 bp and a GC content of 50.7%. A total of 15,407 total protein-coding genes (PCGs) were predicted and functionally annotated. Notably, 857 carbohydrate-active enzymes (CAZymes) were identified in D. eres P3-1W, with 521 CWDEs consisting of 374 glycoside hydrolases (GHs), 108 carbohydrate esterase (CEs) and 91 polysaccharide lyases (PLs). Additionally, 221 auxiliary activities (AAs), 91 glycosyltransferases (GTs), and 108 carbohydrate binding modules (CBMs) were detected. These findings offer valuable insights into the CAZymes of D. eres P3-1W.

Utilizing Deep Neural Networks to Fill Gaps in Small Genomes.

With the widespread adoption of next-generation sequencing technologies, the speed and convenience of genome sequencing have significantly improved, and many biological genomes have been sequenced. However, during the assembly of small genomes, we still face a series of challenges, including repetitive fragments, inverted repeats, low sequencing coverage, and the limitations of sequencing technologies. These challenges lead to unknown gaps in small genomes, hindering complete genome assembly. Although there are many existing assembly software options, they do not fully utilize the potential of artificial intelligence technologies, resulting in limited improvement in gap filling. Here, we propose a novel method, DLGapCloser, based on deep learning, aimed at assisting traditional tools in further filling gaps in small genomes. Firstly, we created four datasets based on the original genomes of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora crassa, and Micromonas pusilla. To further extract effective information from the gene sequences, we also added homologous genomes to enrich the datasets. Secondly, we proposed the DGCNet model, which effectively extracts features and learns context from sequences flanking gaps. Addressing issues with early pruning and high memory usage in the Beam Search algorithm, we developed a new prediction algorithm, Wave-Beam Search. This algorithm alternates between expansion and contraction phases, enhancing efficiency and accuracy. Experimental results showed that the Wave-Beam Search algorithm improved the gap-filling performance of assembly tools by 7.35%, 28.57%, 42.85%, and 8.33% on the original results. Finally, we established new gap-filling standards and created and implemented a novel evaluation method. Validation on the genomes of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora crassa, and Micromonas pusilla showed that DLGapCloser increased the number of filled gaps by 8.05%, 15.3%, 1.4%, and 7% compared to traditional assembly tools.

A high-quality genome assembly and annotation of Thielaviopsis punctulata DSM102798.

Black scorch disease (BSD), caused by the fungal pathogen Thielaviopsis punctulata (Tp) DSM102798, poses a significant threat to date palm cultivation in the United Arab Emirates (UAE). In this study, Chicago and Hi-C libraries were prepared as input for the Dovetail HiRise pipeline to scaffold the genome of Tp DSM102798. We generated an assembly with a total length of 28.23 Mb comprising 1,256 scaffolds, and the assembly had a contig N50 of 18.56 kb, L50 of three, and a BUSCO completeness score of 98.6% for 758 orthologous genes. Annotation of this assembly produced 7,169 genes and 3,501 Gene Ontology (GO) terms. Compared to five other Thielaviopsis genomes, Tp DSM102798 exhibited the highest continuity with a cumulative size of 27.598 Mb for the first seven scaffolds, surpassing the assemblies of all examined strains. These findings offer a foundation for targeted strategies that enhance date palm resistance against BSD, and foster more sustainable and resilient agricultural systems.

Comparative genomic analysis and optimization of astaxanthin production of Rhodotorula paludigena TL35-5 and Rhodotorula sampaioana PL61-2.

Astaxanthin is a powerful antioxidant known to enhance skin, cardiovascular, eye, and brain health. In this study, the genome insights and astaxanthin production of two newly isolated astaxanthin-producing yeasts (TL35-5 and PL61-2) were evaluated and compared. Based on their phenotypic and genotypic characteristics, TL35-5 and PL61-2 were identified as basidiomycetous yeasts belonging to Rhodotorula paludigena and Rhodotorula sampaioana, respectively. To optimize astaxanthin production, the effects of cultural medium composition and cultivation conditions were examined. The optimal conditions for astaxanthin production in R. paludigena TL35-5 involved cultivation in AP medium containing 10 g/L glucose as the sole carbon source, supplemented with 1.92 g/L potassium nitrate, pH 6.5, and incubation at 20°C for 3 days with shaking at 200 rpm. For R. sampaioana PL61-2, the optimal medium composition for astaxanthin production consisted of AP medium with 40 g/L glucose, supplemented with 0.67 g/L urea, pH 7.5, and the fermentation was carried out at 20°C for 3 days with agitating at 200 rpm. Under their optimal conditions, R. paludigena TL35-5 and R. sampaioana PL61-2 gave the highest astaxanthin yields of 3.689 ± 0.031 and 4.680 ± 0.019 mg/L, respectively. The genome of TL35-5 was 20,982,417 bp in length, with a GC content of 64.20%. A total of 6,789 protein-encoding genes were predicted. Similarly, the genome of PL61-2 was 21,374,169 bp long, with a GC content of 64.88%. It contained 6,802 predicted protein-encoding genes. Furthermore, all essential genes involved in astaxanthin biosynthesis, including CrtE, CrtYB, CrtI, CrtS, and CrtR, were identified in both R. paludigena TL35-5 and R. sampaioana PL61-2, providing evidence for their ability to produce astaxanthin.

The loci of environmental adaptation in a model eukaryote.

While the underlying genetic changes have been uncovered in some cases of adaptive evolution, the lack of a systematic study prevents a general understanding of the genomic basis of adaptation. For example, it is unclear whether protein-coding or noncoding mutations are more important to adaptive evolution and whether adaptations to different environments are brought by genetic changes distributed in diverse genes and biological processes or concentrated in a core set. We here perform laboratory evolution of 3360 Saccharomyces cerevisiae populations in 252 environments of varying levels of stress. We find the yeast adaptations to be primarily fueled by large-effect coding mutations overrepresented in a relatively small gene set, despite prevalent antagonistic pleiotropy across environments. Populations generally adapt faster in more stressful environments, partly because of greater benefits of the same mutations in more stressful environments. These and other findings from this model eukaryote help unravel the genomic principles of environmental adaptation.

Genome editing tools based improved applications in macrofungi.

Macrofungi commonly referred to as Mushrooms are distributed worldwide and well known for their nutritional, medicinal, and organoleptic properties. Strain improvement in mushrooms is lagging due to paucity of efficient genome modification techniques. Thus, for advanced developments in research and commercial or economical viability and benefit, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) emerged as an efficient genome editing tool. The higher efficiency and precision of the desired genetic modification(s) are the most valuable attributes of this recent technology. The present review comprehensively summarizes various conventional methods utilized for strain improvement in mushrooms including hybridization, protoplast fusion, and di-mon mating. Furthermore, the problems associated with these techniques have been discussed besides providing the potential recluses. The significance of CRISPR/Cas9 strategies employed for improvement in various mushroom genera has been deliberated, as these strategies will paves the way forward for obtaining improved strain and effective cultivation methods for enhancing the yield and quality of the fruit bodies.

Assembly and comparative analyses of the Geosiphon pyriformis metagenome.

Geosiphon pyriformis, a representative of the fungal sub-phylum Glomeromycotina, is unique in its endosymbiosis with cyanobacteria within a fungal cell. This symbiotic relationship occurs in bladders containing nuclei of G. pyriformis, Mollicutes-like bacterial endosymbionts (MRE), and photosynthetically active and dividing cells of Nostoc punctiforme. Recent genome analyses have shed light on the biology of G. pyriformis, but the genome content and biology of its endosymbionts remain unexplored. To fill this gap, we gathered and examined metagenomic data from the bladders of G. pyriformis, where N. punctiforme and MRE are located. This ensures that our analyses are focused on the organs directly involved in the symbiosis. By comparing this data with the genetic information of related cyanobacteria and MREs from other species of Arbuscular Mycorrhizal Fungi, we aimed to reveal the genetic content of these organisms and understand how they interact at a genetic level to establish a symbiotic relationship. Our analyses uncovered significant gene expansions in the Nostoc endosymbiont, particularly in mobile elements and genes potentially involved in xenobiotic degradation. We also confirmed that the MRE of Glomeromycotina are monophyletic and possess a highly streamlined genome. These genomes show dramatic differences in both structure and content, including the presence of enzymes involved in environmental sensing and stress response.

High phenotypic and genotypic plasticity among strains of the mushroom-forming fungus Schizophyllum commune.

Schizophyllum commune is a mushroom-forming fungus notable for its distinctive fruiting bodies with split gills. It is used as a model organism to study mushroom development, lignocellulose degradation and mating type loci. It is a hypervariable species with considerable genetic and phenotypic diversity between the strains. In this study, we systematically phenotyped 16 dikaryotic strains for aspects of mushroom development and 18 monokaryotic strains for lignocellulose degradation. There was considerable heterogeneity among the strains regarding these phenotypes. The majority of the strains developed mushrooms with varying morphologies, although some strains only grew vegetatively under the tested conditions. Growth on various carbon sources showed strain-specific profiles. The genomes of seven monokaryotic strains were sequenced and analyzed together with six previously published genome sequences. Moreover, the related species Schizophyllum fasciatum was sequenced. Although there was considerable genetic variation between the genome assemblies, the genes related to mushroom formation and lignocellulose degradation were well conserved. These sequenced genomes, in combination with the high phenotypic diversity, will provide a solid basis for functional genomics analyses of the strains of S. commune.